Apparatus for amperometric Diagnostic analysis

ABSTRACT

The present invention relates to a novel method and apparatus for the amperometric determination of an analyte, and in particular, to an apparatus for amperometric analysis utilizing a novel disposable electroanalytical cell for the quantitative determination of biologically important compounds from body fluids.

FIELD OF THE INVENTION

The present invention relates to a disposable electro-analytical celland a method and apparatus for quantitatively determining the presenceof biologically important compounds such as glucose; hormones,therapeutic drugs and the like from body fluids.

Although the present invention has broad applications, for purposes ofillustration of the invention specific emphasis will be placed upon itsapplication in quantitatively determining the presence of a biologicallyimportant compound--glucose.

With Respect To Glucose

Diabetes, and specifically diabetes mellitus, is a metabolic diseasecharacterized by deficient insulin production by the pancreas whichresults in abnormal levels of blood glucose. Although this diseaseafflicts only approximately 4% of the population in the United States,it is the third leading cause of death following heart disease andcancer. With proper maintenance of the patient's blood sugar throughdaily injection of insulin, and strict control of dietary intake, theprognosis for diabetics is excellent. The blood glucose levels must,however, be closely followed in the patient either by clinicallaboratory analysis or by daily analyses which the patient can conductusing relatively simple, non-technical, methods.

At present, current technology for monitoring blood glucose is basedupon visual or instrumental determination of color change produced byenzymatic reactions on a dry reagent pad on a small plastic strip. Thesecolorimetric methods, which utilize the natural oxidant of glucose togluconic acid, specifically oxygen, are based upon the reactions:

    B-D-Glucose+O.sub.2 +H.sub.2 O→D-Gluconic Acid+H.sub.2 O.sub.2

    H.sub.2 O.sub.2 +Reagent→H.sub.2 O+color

Wherein glucose oxidase catalyzes the conversion of B-D Glucose toD-Gluconic Acid. The hydrogen peroxide produced is measured byreflectance spectroscopic methods by its reaction with various dyes, inthe presence of the enzyme peroxidase, to produce a color that ismonitored.

While relatively easy to use, these tests require consistent usertechnique in order to yield reproducible results. For example, thesetests require the removal of blood from a reagent pad at specified andcritical time intervals. After the time interval, excess blood must beremoved by washing and blotting, or by blotting alone, since the colormeasurement is taken at the top surface of the reagent pad. Colordevelopment is either read immediately or after a specified timeinterval.

These steps are dependent upon good and consistent operating techniquerequiring strict attention to timing. Moreover, even utilizing goodoperating technique, calorimetric methods for determining glucose, forexample, have been shown to have poor precision and accuracy,particularly in the hypoglycemic range. Furthermore, instruments usedfor the quantitative calorimetric measurement vary widely in theircalibration methods: some provide no user calibration while othersprovide secondary standards.

Because of the general lack of precision and standardization of thevarious methods and apparatus presently available to test forbiologically important compounds in body fluids, some physicians arehesitant to use such equipment for monitoring levels or dosage. They areparticularly hesitant in recommending such methods for use by thepatients themselves. Accordingly, it is desirable to have a method andapparatus which will permit not only physician but patient self-testingof such compounds with greater reliability.

The present invention addresses the concerns of the physician byproviding enzymatic amperometry methods and apparatus for monitoringcompounds within whole blood, serum, and other body fluids. Enzymaticamperometry provides several advantages for controlling or eliminatingoperator dependant techniques as well as providing a greater lineardynamic range. A system based on this type of method could address theconcerns of the physician hesitant to recommend self-testing for hispatients.

Enzymatic amperometry methods have been applied to the laboratory basedmeasurement of a number of analytes including glucose, blood ureanitrogen, and lactate. Traditionally the electrodes in these systemsconsist of bulk metal wires, cylinders or disks imbedded in aninsulating material. The fabrication process results in individualisticcharacteristics for each electrode necessitating calibration of eachsensor. These electrodes are also too costly for disposable use,necessitating meticulous attention to electrode maintenance forcontinued reliable use. This maintenance is not likely to be performedproperly by untrained personnel (such as patients); therefore, to besuccessful, an enzyme amperometry method intended for self-testing (ornon-traditional site testing) must be based on a disposable sensor thatcan be produced in a manner that allows it to give reproducible outputfrom sensor to sensor and at a cost well below that of traditionalelectrodes.

The present invention addresses these requirements by providingminiaturized disposable electroanalytic sample cells for precisemicro-aliquot sampling, a self-contained, automatic means for measuringthe electrochemical reduction of the sample, and a method for using thecell and apparatus according to the present invention.

The disposable cells according to the present invention are preferablylaminated layers of metallized plastic and nonconducting material. Themetallized layers provide the working and reference electrodes, theareas of which are reproducibly defined by the lamination process. Anopening through these layers is designed to provide thesample-containing area or cell for the precise measurement of thesample. The insertion of the cell into the apparatus according to thepresent invention, automatically initiates the measurement cycle.

To better understand the process of measurement, a presently preferredembodiment of the invention is described which involves a two-stepreaction sequence utilizing a chemical oxidation step using otheroxidants than oxygen, and an electro-chemical reduction step suitablefor quantifying the reaction product of the first step. One advantage toutilizing an oxidant other than dioxygen for the direct determination ofan analyte is that such other oxidants may be prepositioned in thesensor in a large excess of the analyte and thus ensure that the oxidantis not the limiting reagent (with dioxygen, there is normallyinsufficient oxidant initially present in the sensor solution for aquantitative conversion of the analyte).

In the oxidation reaction, a sample containing glucose, for example, isconverted to gluconic acid and a reduction product of the oxidant. Thischemical oxidation reaction has been found to precede to completion inthe presence of an enzyme, glucose oxidase, which is highly specific forthe substrate B-D-glucose, and catalyzes oxidations with single anddouble electron acceptors. It has been found, however, that theoxidation process does not proceed beyond the formation of gluconicacid, thus making this reaction particularly suited for theelectrochemical measurement of glucose.

In a presently preferred embodiment, oxidations with one electronacceptor using ferrocyanide, ferricinum, cobalt (III)orthophenanthroline, and cobalt (III) dipyridyl are preferred.Benzoquinone is a two electron acceptor which also provides excellentelectro-oxidation characteristics for amperometric quantitation.

Amperometric determination of glucose, for example, in accordance withthe present invention utilizes Cottrell current micro-chronoamperometryin which glucose plus an oxidized electron acceptor produces gluconicacid and a reduced acceptor. This determination involves a precedingchemical oxidation step catalyzed by a bi-substrate bi-product enzymaticmechanism as will become apparent throughout this specification.

In this method of quantification, the measurement of a diffusioncontrolled current at one or more accurately specified times (e.g., 5,10, or 15 seconds) after the instant of application of a potential hasthe applicable equation for amperometry at a controlled potential(E=constant) of: ##EQU1## which may also be expressed as:

    i(t)=nFAC.sub.metabolite (D).sup.0.5 (πt).sup.-0.5

where i denotes current, nF is the number of coulombs per mole, A is thearea of the electrode, D is the diffusion coefficient of the reducedform of the reagent, t is the preset time at which the current ismeasured, and C is the concentration of the metabolite. Measurements bythe method according to the present invention of the current due to thereoxidation of the acceptors were found to be proportional to theglucose concentration in the sample.

The method and apparatus of the present invention permit, in preferredembodiments, direct measurements of blood glucose, cholesterol and thelike. Furthermore, the sample cell according to the present invention,provides the testing of controlled volumes of blood withoutpremeasuring. Insertion of the sample cell into the apparatus thuspermits automatic functioning and timing of the reaction allowing forpatient self-testing with a very high degree of precision and accuracy.

One of many of the presently preferred embodiments of the invention foruse in measuring B-D glucose is described in detail to better understandthe nature and scope of the invention. In particular, the method andapparatus according to this embodiment are designed to provide clinicalself-monitoring of blood glucose levels by a diabetic patient. Thesample cell of the invention is used to control the sampling volume andreaction media and acts as the electrochemical sensor. In this describedembodiment, benzoquinone is used as the electron acceptor.

The basic chemical binary reaction utilized by the method according toone preferred embodiment of the present invention is:

    B-D-glucose+Benzoquinone+H.sub.2 O→Gluconic Acid→Hydroquinone

    Hydroquinone→benzoquinone-2e-+2H+.

The first reaction is an oxidation reaction which proceeds to completionin the presence of the enzyme glucose oxidase. Electrochemical oxidationtakes place in the second part of the reaction and provides the meansfor quantifying the amount of hydroquinone produced in the oxidationreaction. This holds true whether catalytic oxidation is conducted withtwo-electron acceptors or one electron acceptor such as ferricyanide[wherein the redox couple would be Fe(CN)₆ ⁻³ /Fe (CN)₆ ⁻⁴ ],ferricinium, cobalt III orthophenanthroline and cobalt (III) dipyridyl.

Catalytic oxidation by glucose oxidase is highly specific forB-D-glucose, but is nonselective as to the oxidant. It has now beendiscovered that the preferred oxidants described above have sufficientlypositive potentials to convert substantially all of the B-D-glucose togluconic acid. Furthermore, this system provides a means by whichamounts as small as 1 mg of glucose (in the preferred embodiment) to1000 mg of glucose can be measured per deciliter of sample--resultswhich have not previously been obtained using other glucose self-testingsystems.

The sensors containing the chemistry to perform the desireddetermination, constructed in accordance with the present invention, areused with a portable meter for self-testing systems. In use, the sensoris inserted into the meter, which turns the meter on and initiates await for the application of the sample. The meter recognizes sampleapplication by the sudden charging current flow that occurs when theelectrodes and the overlaying reagent layer are initially wetted by thesample fluid. Once the sample application is detected, the meter beginsthe reaction incubation step (the length of which is chemistrydependent) to allow the enzymatic reaction to reach completion. Thisperiod is on the order of 15 to 90 seconds for glucose, with incubationtimes of 20 to 45 seconds preferred. Following the incubation period,the instrument then imposes a known potential across the electrodes andmeasures the resulting diffusion limited (i.e., Cottrell) current atspecific time points during the Cottrell current decay. Currentmeasurements can be made in the range of 2 to 30 seconds followingpotential application with measurement times of 10 to 20 secondspreferred. These current values are then used to calculate the analyteconcentration which is then displayed. The meter will then wait foreither the user to remove the sensor or for a predetermined periodbefore shutting itself down.

Due to the nature of the Cottrell current, it is possible to develop acalibration curve at more than one time point following application ofthe potential in order to verify that the measurement is beingaccurately made. Results can then be calculated at the different timepoints and compared. This is illustrated schematically in FIGS. 11 and12; which indicate expected, or "normal" Cottrell curves, A, B, C, D,for various glucose concentrations and an abnormal curve E, showingdivergence from expected curve D as indicated by the multiple currentreadings. In a system that is operating correctly, the results shouldagree within reasonable limits. The exact range of acceptable differencebetween the expected and measured currents depends on a number ofcompromises but would generally be in the range of 1-10%. Resultsoutside of the acceptable limits would indicate some problem with thesystem. For instance, incomplete wetting of the reagent (i.e., too smallof a drop of blood) would result in failure to follow the Cottrell curvedecay and result in a higher value being calculated at subsequentmeasurement points than would have been expected for Cottrell currentcurve delay. FIG. 13 represents a schematic circuit diagram which can beemployed in producing a preferred embodiment of the invention for takingmultiple current measurements.

The present invention provides for a measurement system that eliminatesseveral of the critical operator dependant variables that adverselyaffect the accuracy and reliability and provides for a greater dynamicrange than other self-testing systems.

These and other advantages of the present invention will become apparentfrom a perusal of the following detailed description of one embodimentpresently preferred for measuring glucose and other analytes which is tobe taken in conjunction with the accompanying drawings in which likenumerals indicate like components and in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded view of a portable testing apparatus according tothe present invention;

FIG. 2 is a plan view of the sampling cell of the present invention;

FIG. 3 is an exploded view of the sample cell shown in FIG. 2;

FIG. 4 is an exploded view of another embodiment of a sample cellaccording to the invention;

FIG. 5 is a plan view of the cell shown in FIG. 4;

FIG. 6 is a graph showing current as a function of glucoseconcentration;

FIG. 7 is a graphical presentation of Cottrell current as a function ofglucose concentration; and

FIG. 8 is a presently preferred circuit diagram of an electrical circuitfor use in the apparatus shown in FIG. 1.

FIG. 9 is a preferred embodiment of the electrochemical cell of theinvention wherein the reference electrode area is greater than theworking electrode area.

FIG. 10 is a preferred embodiment of the invention wherein the twoelectrodes are co-planar, equal size, and preferably the same noblemetal.

FIG. 11 is a graph showing multiple current measurements of the presentinvention.

FIG. 12 is a graph correlating measured current to glucose concentrationfor the curves of FIG. 11.

FIG. 13 is a schematic circuit diagram depicting a preferred embodimentof the invention.

With specific reference to FIG. 1, a portable electrochemical testingapparatus 10 is shown for use in patient self-testing, such as, forexample, for blood glucose levels. Apparatus 10 comprises a front andback housing 11 and 12, respectively, a front panel 13 and a circuitboard 15. Front panel 13 includes graphic display panels 16 forproviding information and instructions to the patient, and directread-out of the test results. While a start button 18 is provided toinitiate an analysis, it is preferred that the system begin operationwhen a sample cell 20 (FIG. 2) is inserted into the window 19 of theapparatus.

With reference to FIGS. 2 and 3, sample cell 20 is a metallized plasticsubstrate having a specifically-sized opening 21 which defines avolumetric well 21, when the cell is assembled, for containing a reagentpad and the blood to be analyzed. Cell 20 comprises a first substrate 22and a second substrate 23 which may be preferably made from styrene orother substantially non-conducting plastic, such as polyimide,polyethylene, etc. Polyimide has proven particularly preferred becausethe metal adheres well to it, and the electrodes may be more readilyslitted to the desired width. Polyimide sold under the trademark KAPTON,and available from DuPont, has proven particularly advantageous.

Positioned on second substrate 23 is reference electrode 24. Referenceelectrode 24 may be preferably manufactured, for example, by vapordepositing or "sputtering" the electrode onto a substrate made from amaterial such as the polyimide Kapton. In one preferred embodiment,reference electrode 24 is a silver--silver chloride electrode. Thiselectrode can be produced by first depositing a silver chloride layer ona silver layer by either chemical or electrochemical means before thesubstrate is used to construct the cells. The silver chloride layer mayeven be generated in-situ on a silver electrode when the reagent layercontains certain of the oxidants, such as ferricyanide, and chloride asshown in the following reactions:

    Ag+Ox→Ag.sup.- +Red

    Ag.sup.- +Cl.sup.- →AgCl

Alternatively the silver--silver chloride electrode can be produced bydepositing a layer of silver oxide (by reactive sputtering) onto thesilver film. The silver oxide layer is then converted in-situ at thetime of testing to silver chloride according to the reaction:

    Ag.sub.2 O+H.sub.2 O+2Cl.sup.- →2AgCl+2(OH).sup.-

when the sensor is wetted by the sample fluid and reconstitutes thechloride containing reagent layer. The silver electrode is thus coatedwith a layer containing silver chloride.

The reference electrode may also be of the type generally known as a"pseudo" reference electrode which relies upon the large excess of theoxidizing species to establish a known potential at a noble metalelectrode. In a preferred embodiment, two electrodes of the same noblemetal or carbon are used, however one is generally of greater surfacearea and is used as the reference electrode. The large excess of theoxidized species and the larger surface area of the reference resists ashift of the potential of the reference electrode.

The primary requirement for the pseudo-reference (as is also the casewith traditional reference electrodes) is that it should be able tosupply the necessary current (in opposition to the current flow at theworking electrode) without significant shift in its potential. Providinga larger surface area for the reference electrode than for the workingelectrode is one way to accomplish this. When high concentration of theoxidant are utilized and/or the range of currents is kept relativelylow, e.g., less than about 20 to 40 milliamps/cm², with 0.1Mferricyanide as the oxidant, it is possible to reduce the ratio of thereference to working electrode to 1:1 or even less. The use of equalsize electrodes offers some advantage in terms of manufacture but withpotentially a limitation to the upper range.

In the case of same size (i.e., same surface area) electrodes, a largeexcess of oxidized species (i.e., wherein the oxidized form of the redoxmediator (i.e., ferricyanide) is present in the reagent layer insufficient excess to insure that the diffusion limited electrooxidationof the redox mediator at the working electrode surface is the principallimiter of current flow through the cell and resists a shift of thepotential of the second electrode (i.e., pseudo-reference) vis-a-vis thefirst (i.e., working) electrode.

In a highly preferred embodiment of the invention, the two-electrodesystem uses same size, same metal electrodes. Preferably noble metals,such as palladium are used. In this palladium vs. palladium embodiment,a potential of +0.30 volts applied between the electrodes has proveneffective when ferricyanide is the oxidant. Acceptable same-sizepalladium vs. palladium coplanar-electrodes may be produced frommetalized plastic slitted by a high performance slitter such as MetlonCorp., 133 Francis Avenue, Cranston, R.I. 02910.

In the pseudo-reference instance, care must be exercised in the spacingof the electrodes. This is due to the fact that as the second electrodefunctions to provide the balancing current, it is actually producing thereduced form of the oxidant (redox mediator). If the electrodes arespaced too closely together, the reduced form produced at the secondelectrode can diffuse toward the working electrode, where it would thenbe re-oxidized, adding to the current flow due to the oxidation of thereduced form produced by the enzymatic reaction. The net effect would bea departure from the expected Cottrell current decay curve illustratedin FIGS. 7 and 11.

This problem is potentially aggravated when the size of the secondelectrode, vis-a-vis the first, is reduced, such as in the case ofsame-size electrodes or when the second electrode is smaller than theworking electrode. This is due to the higher effective concentration ofreduced oxidant produced over the surface of the second electrode. Thiscan be shown by the following analysis. The current flow at the twoelectrodes can be represented by the Cottrell equation. For the firstelectrode, that is: ##EQU2## At the second electrode that is: ##EQU3##Since the two currents must be of the same magnitude (but oppositesign), the following is true at any given time:

    dC.sub.1st A.sub.1st =dC.sub.2nd A.sub.2nd

Where A_(1st) is the area of the first (i.e., working) electrode,C_(1st) is the concentration of the reduced form of oxidant in thesolution in the reagent layer at time zero (the instant the potential isapplied), A_(2nd) is the area of the second (i.e., reference or counter)electrode, and C_(2nd) is the concentration of reduced form of theoxidant generated at or by the second electrode at time zero (theinstant the potential is applied).

Since the working electrode is poised to control the current flow, thesecond electrode acts to balance current flow. Thus, the smaller thesecond electrode, the higher the concentration of the reduced oxidantproduced at the surface of that electrode; the higher the concentration,the greater the diffusion gradient and the greater the potential for thereduced oxidant produced at the second electrode to diffuse towards theworking electrode and cause an undesired additional current flow,resulting in a departure from expected Cottrell current flow for thesystem. First and second electrodes 424, 426 must be spaced apart by adistance d (FIG. 10) sufficient to avoid this departure from expectedCottrell current. A distance d of at least 0.1 mm, preferably greaterthan 0.1 mm, has proven effective at current flows of 0-50 microamps andabout 5 mm² working electrode area. The higher the expected currentdensity, the greater d must be in order to insure true Cottrellmeasurement. Also, if measurement times exceed about 30 seconds, it isnecessary to increase the distance d. In a highly preferred embodimentof the invention, d is about 1-2 mm, A_(1st) and A_(2nd) are both about5 mm², current flow is about 0-100 microamps, with measurement time ofless than 30 seconds.

The maximum spacing for d is dictated by the conductivity of thesolution, which effects to the actual voltage at the electrodes(obviated in 3-electrode systems). Practical considerations, however,dictate that the spacing d be as small as technically feasible tominimize substrate, reagent, and sample size requirements. Ideally, thepreferred sample is the size of a drop of blood of less than 20microliters, which must bridge the electrodes and wet the reagent layer.As a practical matter, the spacing d should not exceed about 1 cmbecause of the difficulty of bridging such a gap with a drop of blood ofthis size and substantially instantaneously spreading that drop of bloodacross the reagent layer. It is important that the sample, i.e., blood,spread across the reagent layer substantially instantaneously followingapplication of the sample to the cell, in order to avoid migration ofreagents, or erosion of reagents from the site at which the drop ofblood was applied. The wicking layer is helpful in this regard.

EXAMPLE 1

A two mm² palladium indicator electrode was referenced to a two mm²Ag/Ag₂ O electrode and the electrodes were spaced apart by a 2 mm gap.Measurements were taken from sample cells including the reagentsdescribed herein, including an analyte (i.e., glucose) the oxidized formof a redox mediator (i.e., ferricyanide), an enzyme (i.e., glucoseoxidase), and a buffer (i.e., phosphate). Preferably, in the case ofsame size, same noble metal electrodes, the oxidized form of the redoxmediator is present in sufficient quantity to insure that the counterelectrode current, i.e., that produced at the reference electrode, isnot limiting as a result of conversion to the reduced form of the redoxmediator at the working electrode surface. The data for the results,summarized in Table 1, were obtained 10 seconds after current wasapplied.

For 5 millimolar ferrocyanide corresponding to 4 millimolar glucose (72mg/dl), an average current of 27.1 microamps was obtained. The relativestandard deviation was 5.3%. For 20 millimolar ferrocyanide, an averagecurrent of 102.9 was obtained and the relative standard deviation was3.9%. In this experiment, the volumes tested were 50 microliters,although smaller volumes such as 10 microliters could be employed if thearea of the 2 mm² electrode strips were decreased.

A concentration series for ferrocyanide from 0 through 30 millimolar wassimilarly obtained. A plot of the currents at 10 seconds vs.ferrocyanide concentration indicated a linearly increasing current withconcentration through 25 millimolar (approximately 360 mg/dl glucose).Similar results can be obtained when both same-size electrodes are thesame noble metal, such as palladium.

The buffer used in the reagent layer is preferably non-reactive withrespect to the reduced and oxidized form of the redox mediator (i.e.,has a higher oxidation potential). Phosphate buffer has proven effectivein this regard, although other suitable buffers would, of course, now bereadily apparent to those of ordinary skill in the art.

                  TABLE 1                                                         ______________________________________                                        Cottrell Current, microamps,                                                                  Fe (CN).sub.6.sup.4-                                                                     Corresponding                                      at 10 seconds after                                                                           Concentration,                                                                           Glucose                                            E is applied    millimolar Concentration, mg/dl                               ______________________________________                                        0               0          0                                                  27              5          72                                                 58              10         144                                                75              15         216                                                103             20         288                                                142             25         360                                                136             30         432                                                ______________________________________                                    

Indicator or working electrode 26 can be either a strip of platinum,gold, or palladium metallized plastic positioned on reference electrode24, or, alternately, as showned in FIG. 10, the working electrode 426and reference electrode 424 are laminated between an upper 422 and lower423 non-conducting (i.e. electrically insulating) material, such aspolyethylene or polystyrene. Preferably, sample cell 20 is prepared bysandwiching or laminating the electrodes between the substrate to form acomposite unit. Of course, other methods of applying the metal or otherelectrically conducting material, such as, without limitation, silkscreening, vapor deposition, electrolysis, adhesion, etc., may also beemployed.

Of course, any combination of suitable electrode pairs may be used.Names other than "working" vs. "reference" electrodes which have beeninterchangeably used in electrochemical applications include, "working"v.s. "counter" electrodes, "excitation" v. "source" electrodes, or threeelectrode systems having a "working" electrode, a "reference" electrode,and an "auxiliary" electrode. In the case of a three-electrode system,the auxiliary electrode completes the circuit, allowing current to flowthrough the cell, while the reference electrode maintains a constantinterfacial potential difference regardless of the current. In the twoelectrode scenario, the second electrode serves both the function of anauxiliary and reference electrode. Regardless of the nomenclatureemployed, the electrochemistry of interest in the present inventionoccurs at a first electrode (i.e., working) and a second electrode(i.e., reference or counter) acts to counter balance current flow (i.e.,provide opposing current flow to the first electrode) and fix theoperating potential of the system.

As shown in FIG. 2, first substrate 22 is of a slightly shorter lengthso as to expose an end portion 27 of electrodes 24 and 26 and allow forelectrical contact with the testing circuit contained in the apparatus.In this embodiment, after a sample has been positioned within well 21,cell 20 is pushed into window 19 of the front panel to initiate testing.In this embodiment, a reagent may be applied to well 21, or, preferably,a pad of dry reagent is positioned therein and a sample (drop) of bloodis placed into the well 21 containing the reagent.

Referring to FIGS. 4-5, alternative embodiments of sample cell 20 areshown. In FIG. 4, sample cell 120 is shown having first 122 and second123 substrates. Reference electrode 124 and working electrode 126 arelaminated between substrates 122 and 123. Opening 121 is dimensioned tocontain the sample for testing. End 130 (FIG. 5) is designed to beinserted into the apparatus, and electrical contact is made with therespective electrodes through cut-outs 131 and 132 on the cell.Reference electrode 124 also includes cut out 133 to permit electricalcontact with working electrode 126.

Referring to FIGS. 1 and 2, the sample cell according to the presentinvention is positioned through window 19 (FIG. 1) to initiate thetesting procedure. Once inserted, a potential is applied at portion 27(FIG. 2) of the sample cell across electrodes 24 and 26 to detect thepresence of the sample. Once the sample's presence is detected, thepotential is removed and the incubation period initiated. Optionallyduring this period, a vibrator means 31 (FIG. 1) may be activated toprovide agitation of the reagents in order to enhance dissolution (anincubation period of 20 to 45 seconds is conveniently used for thedetermination of glucose and no vibration is normally required). Anelectrical potential is next applied at portion 27 of the sample cell toelectrodes 24 and 26 and the current through the sample is measured anddisplayed on display 16.

To fully take advantage of the above apparatus, the needed chemistry forthe self testing systems is incorporated into a dry reagent layer thatis positioned onto the disposable cell creating a complete sensor forthe intended analyte. The disposable electrochemical cell is constructedby the lamination of metallized plastics and nonconducting materials insuch a way that there is a precisely defined working electrode area. Thereagent layer is either directly coated onto the cell or preferablyincorporated (coated) into a supporting matrix such as filter paper,membrane filter, woven fabric or non-woven fabric, which is then placedinto the cell, substantially covering the electrode surfaces exposed bythe cutout portion or window of the electrically insulating upperlaminate. When a supporting matrix is used, its pore size and voidvolume can be adjusted to provide the desired precision and mechanicalsupport. In general, membrane filters or nonwoven fabrics provide thebest materials for the reagent layer support. Pore sizes of 0.45 to 50um and void volumes of 50-90% are appropriate. The coating formulationgenerally includes a binder such as gelatin, carrageenan,methylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc., thatacts to delay the dissolution of the reagents until the reagent layerhas absorbed most of the fluid from the sample. The concentration of thebinder is generally on the order of 0.1 to 10% with 1-4% preferred.

The reagent layer imbibes a fixed amount of the sample fluid when it isapplied to the surface of the layer thus eliminating any need forpremeasurement of sample volume. Furthermore, by virtue of measuringcurrent flow rather than reflected light, there is no need to remove theblood from the surface of the reagent layer prior to measurement asthere is with reflectance spectroscopy systems. While the fluid samplecould be applied directly to the surface of the reagent layer, tofacilitate spread of blood across the entire surface of the reagentlayer the sensor preferably includes a dispersing spreading or wickinglayer. This layer, generally a nonwoven fabric or adsorbent paper, ispositioned over the reagent layer and acts to rapidly distribute theblood over the reagent layer. In some applications this dispersing layercould incorporate additional reagents.

For glucose determination, cells utilizing the coplanar design wereconstructed having the reagent layer containing the followingformulations:

    ______________________________________                                        Glucose oxidase       600    units/ml                                         Potassium Ferricyanide                                                                              0.4    M                                                Phosphate Buffer      0.1    M                                                Potassium Chloride    0.5    M                                                Gelatin               2.0    g/dl                                             ______________________________________                                    

This was produced by coating a membrane filter with a solution of theabove composition and air drying. The reagent layer was then cut intostrips that just fit the window opening of the cells and these stripswere placed over the electrodes exposed within the windows. A wickinglayer of a non-woven rayon fabric was then placed over this reagentlayer and held in place with an overlay tape.

As will now be readily appreciated by those of ordinary skill in theart, the enzyme (i.e., glucose oxidase) is sufficient in type and amountto catalyze the reaction (i.e., receive at least one electron from thereaction) involving the enzyme, the analyte (i.e., glucose) and theoxidized form of the redox mediator (i.e., ferricyanide). Optionally,surfactant can also be used in the reagent layer to promote wetting ofthe reagent by the sample containing the analyte.

In order to prove the application of the technology according to thepresent invention, a large number of examples were run in aqueoussolution at 25° C. The electrolyte consisted of a phosphate buffer of pH6.8 which was about 0.1 molar total phosphate and 0.5M potassiumchloride reagent. The potentials are referenced to a normal hydrogenelectrode (NHE). In these tests it was found that any potential betweenapproximately +0.8 and 1.2 volt (vs NHE) is suitable for thequantification of hydroquinone when benzoquinone is used as the oxidant.The limiting currents are proportional to hydroquinone concentrations inthe range between 0.0001M and 0.050M.

Determination of glucose by Cottrell current (i_(t))microchronoamperometry with the present method is created in thereaction of hydroquinone to benzoquinone. Cottrell currents decay withtime in accordance with the equation:

    .sup.i r.sup.t1/2 =const

where t denotes time.

The main difference between these two techniques consists of applyingthe appropriate controlled potential after the glucose-benzoquinonereaction is complete and correlating glucose concentrations withCottrell currents measured at a fixed time thereafter. The current-timereadout is shown in FIG. 7. Proportionality between glucoseconcentrations and Cottrell currents (recorded at t=30 seconds after theapplication of potential) is shown in FIG. 6.

It should be noted that Cottrell chronoamperometry of metabolites needsthe dual safeguards of enzymatic catalysis and controlled potentialelectrolysis. Gluconic acid yields of 99.9+ percent were attained in thepresence of glucose oxidase. Concomitantly, equivalent amounts ofbenzoquinone were reduced to hydroquinone, which was convenientlyquantitated in quiescent solutions, at stationary palladium thin filmanodes or sample cells.

The results of these many tests demonstrates the microchronoamperometricmethodology of the present invention and its practicality for glucoseself-monitoring by diabetics.

In a presently preferred embodiment of the invention utilizingferricyanide, a number of tests were run showing certain improvedoperating capabilities.

Referring to FIG. 8, a schematic diagram of a preferred circuit 15 foruse in the apparatus 10 is shown. Circuit 15 includes a microprocessorand LCD panel 16. The working and reference electrodes on the samplecell 20 make contact at contacts W (working electrode) and R (referenceelectrode), respectively. Voltage reference 41 is connected to battery42 through analogue power switch 43. Current from electrodes W and R isconverted to a voltage by op amp 45. That voltage is converted into adigital signal (frequency) by a voltage to frequency converter 46electrically connected to the microprocessor 48. The microprocessor 48controls the timing of the signals. Measurement of current flow isconverted by microprocessor 48 to equivalent glucose, cholesterol orother substance concentrations. Other circuits within the skills of apracticed engineer can now be utilized to obtain the advantages of thepresent invention.

With regard to FIG. 9, cell 400 consists of coplanar first (i.e.,working) 426 and second (i.e., reference) 424 electrodes laminatedbetween an upper 422 and lower 423 nonconducting (i.e., electricallyinsulating) material. Lamination is on an adhesive layer 425. The uppermaterial 422 includes a die cut opening 428 which, along with the widthof the working electrode material defines the working electrode area andprovides (with an overlapping reagent layer not depicted) the samplingport of the cell. As illustrated, this die-cut opening is rectangular orsquare, which has proven advantageous from the standpoint ofreproduceability, as such openings are more readily centered over theco-planar electrodes than circular openings. At one end of cell 400 isan open area 427 similar to end position 27 of FIG. 2. In the case ofsame size (i.e., same surface area first and second electrodes 426, 424as illustrated in FIG. 10), the opening 428 insures that equal surfaceareas of the first and second electrodes are exposed.

The efficiency of using the apparatus according to the present inventionto provide a means for in-home self testing by patients such asdiabetics (in the preferred embodiment) can be seen in the followingtable in which the technology according to the present invention iscompared to four commercially available units. As will be seen, thepresent invention is simpler, and in this instance simplicity breedsconsistency in results.

    ______________________________________                                        GLUCOSE SYSTEM COMPARISONS                                                                                            Present                               Steps       1       2       3     4     Invention                             ______________________________________                                        Turn Instrument On                                                                        X       X       X     X     X                                     Calibrate Instrument                                                                      X       X                                                         Finger Puncture                                                                           X       X       X     X     X                                     Apply Blood X       X       X     X     X                                     Initiate Timing                                                                           X       X       X                                                 Sequence                                                                      Blot        X       X       X                                                 Insert Strip to Read                                                                      X       X       X     X                                           Read Results                                                                              X       X       X     X     X                                     Total Steps Per                                                                           8       8       7     5     4                                     Testing                                                                       Detection System                                                                          RS'     RS      RS    RS    Polaro-                                                                       graphic                               Range (mg/dl)                                                                             10-400  40-400  25-450                                                                              40-400                                                                              0-1000                                CV"                                                                           Hypoglycemic                                                                              15%     15%                 5%                                    Euglycemic  10%     10%                 3%                                    Hyperglycemic                                                                              5%      5%                 2%                                    Correlation 0.921   0.862               0.95                                  ______________________________________                                         'RS  Reflectance Spectroscopy                                                 "Coefficient of variation                                                

Thus, while we have illustrated and described the preferred embodimentof our invention, it is to be understood that this invention is capableof variation and modification, and we therefore do not wish or intend tobe limited to the precise terms set forth, but desire and intend toavail ourselves of such changes and alterations which may be made foradapting the present invention to various usages and conditions.Accordingly, such changes and alterations are properly intended to bewithin the full range of equivalents, and therefore within the purview,of the following claims. The terms and expressions which have beenemployed in the foregoing specifications are used therein as terms ofdescription and not of limitation, and thus there is no intention, inthe use of such terms and expressions, of excluding equivalents of thefeatures shown and described or portions thereof, it being recognizedthat the scope of the invention is defined and limited only by theclaims which follow.

Having thus described our invention and the manner and process of makingand using it in such full, clear, concise, and exact terms so as toenable any person skilled in the art to which it pertains, or to withwhich it is most nearly connected, to make and use the same.

We claim:
 1. An apparatus for measuring compounds in a sample fluid,comprising:a) a housing having an access opening therethrough; b) asample cell receivable into said access opening of said housing, saidsample cell being composed of;(i) a first electrode which acts as aworking electrode; (ii) a second electrode which acts to fix the systempotential and provide opposing current flow with respect to said firstelectrode, said second electrode being made of the same electricallyconducting material as said first electrode, and being operativelyassociated with said first electrode, the ratio of the surface area ofsaid second electrode to the surface area of said first electrode being1:1 or less; (iii) at least one non-conducting layer member having anopening therethrough, said at least one non-conducting layer memberbeing disposed in contact with at least one of said first and secondelectrodes and being sealed against at least one of said first andsecond electrodes to form a known electrode area within said openingsuch that said opening forms a well to receive the sample fluid and toallow a user of said apparatus to place the sample fluid in said knownelectrode area in contact with said first electrode and said secondelectrode; c) means for applying an electrical potential to both saidfirst electrode and said second electrode; d) means for creating anelectrical circuit between said first electrode and said secondelectrode through the sample fluid; e) means for measuring a firstCottrell current reading through the sample fluid at a firstpredetermined time after the electrical potential is applied and forobtaining at least one additional Cottrell current reading through thesample fluid, the at least one additional Cottrell current readingoccurring at a second predetermined time following the firstpredetermined time; f) microprocessor means for converting the firstCottrell current reading into a first analyte concentration measurementusing a calibration slope and an intercept specific for the firstCottrell current measurement, for converting the at least one additionalCottrell current reading into an additional analyte concentration usinga calibration slope and an intercept specific for the at least oneadditional Cottrell current measurement, and for comparing the firstanalyte concentration measurement with the at least one additionalconcentration measurement to confirm that they are within a prescribedpercentage of each other; and g) means for visually displaying theresults of said analyte concentration measurements.
 2. The apparatus ofclaim 1, further comprising;means for initiating an electrical potentialupon insertion of the sample fluid to detect the presence of the samplefluid, and said initiation means including means for signaling saidmicroprocessor means to commence a reaction timing sequence when thepresence of the sample fluid is detected, and means for removing thepotential during the reaction timing sequence.
 3. The apparatus of claim1, wherein said first and second electrodes are spaced apart by adistance of at least about 0.1 mm and at most about 1 cm.
 4. A devicefor obtaining measurements of an analyte contained in a sample in orderto determine the concentration of analyte in the sample, said devicecomprising:a) a first electrical insulator; b) a pair of electrodescomprising working and counter electrodes, the working and counterelectrodes being made of the same electrically conducting materials andbeing supported on the first electrical insulator; c) a secondelectrical insulator, overlaying said first electrical insulator andsaid working and counter electrodes and including a cutout portion thatexposes surface areas of said working and counter electrodes; and d) areagent, substantially covering the exposed electrode surfaces in thecutout portion and comprising the oxidized form of a redox mediator, andan enzyme, the oxidized form of the redox mediator being capable ofreceiving at least one electron from a reaction involving enzyme,analyte, and the oxidized form of the redox mediator and being insufficient amount to insure that current produced by diffusion limitedelectrooxidation as a result of the reaction is limited by the oxidationof the reduced form of the redox mediator at the working electrodesurface, the enzyme being present in sufficient amount to catalyze thereaction involving the enzyme, analyte, and the oxidized form of theredox mediator, and said device configured to measure a first Cottrellcurrent attributable to the diffusion limited electrooxidation at afirst predetermined time, and to measure at least one second Cottrellcurrent attributable to the diffusion limited electrooxidation at one ormore subsequent predetermined times, said device including amicroprocessor means for converting the first Cottrell current into afirst analyte concentration measurement using a calibration slope and anintercept specific for the first Cottrell current measurement, and forconverting the at least one second Cottrell current reading into anadditional analyte concentration using a calibration slope and anintercept specific for the at least one second Cottrell currentmeasurement, and for comparing the first analyte concentrationmeasurement with the at least one additional concentration measurementto confirm that they are within a prescribed percentage of each other.5. A system for obtaining measurements of analytes contained in a samplein order to determine the concentration of analyte in the sample, saidsystem comprising reagents incorporated into a sample receiving portionof an electrochemical device that measures the analytes and that has apair of electrodes comprising working and counter electrodes, theworking and counter electrodes being made of the same electricallyconducting materials, the ratio of the surface area of said counterelectrode to the surface area of said working electrode being 1:1 orless, said reagents comprising:a) the oxidized form of a redox mediator,an enzyme, and a buffer,(i) the oxidized form of the redox mediatorbeing capable of receiving at least one electron from a reactioninvolving enzyme, analyte, and the oxidized form of the redox mediatorand being present in sufficient amount to insure that current producedby diffusion limited electrooxidation as a result of the reactionfollowing application of a potential to the working and counterelectrodes is limited by the oxidation of the reduced form of the redoxmediator at the working electrode surface, (ii) the enzyme being presentin sufficient amount to catalyze the reaction involving the enzyme,analyte, and the oxidized form of the redox mediator, and (iii) thebuffer having a higher oxidation potential than the reduced form of theredox mediator and being present in sufficient amount to provide andmaintain a pH at which the enzyme catalyzes the reaction involving theenzyme, analyte, and the oxidized form of the redox mediator, saidsystem configured to measure a first Cottrell current attributable tothe diffusion limited electrooxidation at a first predetermined time,and to measure at least one second Cottrell current attributable to thediffusion limited electrooxidation at one or more subsequentpredetermined times, said system including a microprocessor means forconverting the first Cottrell current into a first analyte concentrationmeasurement using a calibration slope and an intercept specific for thefirst Cottrell current measurement, and for converting the at least onesecond Cottrell current reading into an additional analyte concentrationusing a calibration slope and an intercept specific for the at least onesecond Cottrell current measurement, and for comparing the first analyteconcentration measurement with the at least one additional concentrationmeasurement to confirm that they are within a prescribed percentage ofeach other.
 6. A system for obtaining measurements of analytes containedin a sample in order to determine the concentration of analyte in thesample, said system comprising reagents incorporated into a samplereceiving portion of an electrochemical device that measures an analyteand that has a pair of electrodes comprising working and counterelectrodes, the working and counter electrodes being made of the sameelectrically conducting materials, the ratio of the surface area of saidcounter electrode to the surface area of said working electrode being1:1 or less, said reagents comprising:a) the reduced form of a redoxmediator, an enzyme, and a buffer,(i) the reduced form of the redoxmediator being capable of donating at least one electron from a reactioninvolving enzyme, analyte, and the reduced form of the redox mediatorand being present in sufficient amount to insure that current producedby diffusion limited electroreduction as a result of the reactionfollowing application of a potential to the working and counterelectrodes is limited by the reduction of the oxidized form of the redoxmediator at the working electrode surface, (ii) the enzyme being presentin sufficient amount to catalyze the reaction involving the enzyme,analyte, and the reduced form of the redox mediator, and (iii) thebuffer having a lower reduction potential than the oxidized form of theredox mediator and being present in sufficient amount to provide andmaintain a pH at which the enzyme catalyzes the reaction involving theenzyme, analyte, and the reduced form of the redox mediator, said systemconfigured to measure a first Cottrell current attributable to thediffusion limited electroreduction at a first predetermined time, and tomeasure at least one second Cottrell current attributable to thediffusion limited electroreduction at one or more subsequentpredetermined times, and including microprocessor means for convertingthe first Cottrell current into a first analyte concentrationmeasurement using a calibration slope and an intercept specific for thefirst Cottrell current measurement, and for converting the at least onesecond Cottrell current reading into an additional analyte concentrationusing a calibration slope and an intercept specific for the at least onesecond Cottrell current measurement, and for comparing the first analyteconcentration measurement with the at least one additional concentrationmeasurement to confirm that they are within a prescribed percentage ofeach other.